Insects must recognize pathogens before they can mount an immune response. Recognition of pathogens in insects is mediated by pattern recognition receptors (PRRs) such as C-type lectins. In the tobacco hornworm, Manduca sexta, C-type lectins have been shown to function as stimulatory PRRs to enhance immune responses. In the mosquito Anopheles gambiae, a vector for malaria, and two C-type lectins, CTL4 and CTLMA2, somehow prevent Plasmodium berghei from melanization. But LRIM-1 and TEP-1 can enhance P. berghei melanization. However, silencing of CTL4, CTLMA2 and LRIM-1 in A. gambiae has no effect on development of P. falciparum, the human parasite, due to immune evasion. How can P. falciparum evade the immune system of A. gambiae, while P. berghei can not? We hypothesize that interaction of proteins from A. gambiae plasma or extracellular matrix with the surface of Plasmodium parasites are the keys. This proposal aims to investigate A. gambiae C-type lectins as putative PRRs to modulate immune responses against Plasmodium. The specific aims are: 1. Continue to investigate detailed molecular interactions of an insect prototype C-type lectin, Manduca immulectin-2, as a guide to understanding potential lectin-carbohydrate interactions and lectin-protein interactions that influence the outcome of pattern recognition and innate immune responses. We will construct deletions and point mutations in the carboxyl-terminal carbohydrate recognition domain of immulectin-2 predicted to affect interactions with microbial polysaccharides or with protein components of the prophenoloxidase activation complex. 2. Investigate the ligand-binding specificity and affinity of nine A. gambiae C-type lectins (including CTL4 and CTLMA2) and two chimeric lectins, as well as TEP-1 (thioester-containing protein-1) and LRIM-1 (leucine-rich repeat immune gene-1), study interactions of these proteins with P. berghei ookinetes and ookinete surface proteins, and identify P. berghei ookinete surface proteins that can be recognize by these A. gambiae proteins. 3. Study functions of nine A. gambiae lectins, two chimeric lectins, TEP-1 and LRIM-1 in encapsulation, melanization, phagocytosis and phenoloxidase activation in mosquitoes, and investigate interactions of these proteins during immune responses in A. gambiae. Our long term goal is to understand parasite recognition and parasite-pattern recognition receptor interaction in vector insects. PUBLIC HEALTH RELEVANCE Insects must recognize pathogens and parasites before they can mount an immune response. Recognition of nonself pathogens in insects is mediated by pattern recognition receptors (PRRs). We have some understanding of the proteins that serve as PRRs in insects to stimulate immune responses to bacterial or fungal infection. However, very little is known about how insect immune systems recognize non-fungal eukaryotic parasites: what are the pathogen-associated molecular patterns (PAMPs) on such organisms and what PRRs bind to them? This project is to investigate C-type lectins as potential PRRs in innate immune responses of Anopheles gambiae against Plasmodium parasites.